Project Summary Natural killer (NK) cells are a critical component of innate immunity and are cytolytic to tumor cells and viral- infected cells. Oncolytic herpes simplex virus 1 (oHSV), which has recently been approved by the FDA for the treatment of cancer, holds great potential in the treatment of glioblastoma (GBM), a highly lethal brain tumor. However, based on our previous studies, host NK cells provide a rapid and robust response following central nervous system (CNS) infection with oHSV, thus presenting a barrier for effective oncolytic virotherapy for GBM. Cytotoxic genes, including granzyme B (Gzmb), determine NK cell cytolytic activity in these settings; however, the molecular mechanisms responsible for regulating Gzmb expression are largely unknown. In this application, our preliminary data show that Smad4, a co-Smad protein in the TGF-beta superfamily signaling pathway, positively regulates Gzmb expression in a TGF-beta-independent manner, which correlates with decreased anti-tumor activity in mice with an NK-specific Smad4 deficiency. Using this mouse model, we also discovered that Smad4 positively regulates NK cell homeostasis and maturation by upregulating Blimp1, a positive regulator of NK cell maturation. Smad4 is also a co-Smad in bone morphogenetic protein (BMP) signaling. Our preliminary data support the hypothesis that the TGF-?-independent role of Smad4 in regulating NK cell function is downstream of BMP signaling, which has been reported to positively regulate NK cell function. Here, we propose to explore the mechanisms for this conceptually novel discovery and to modulate BMP-Smad4 signaling in NK cells in the setting of oncolytic virotherapy for GBM. Our overall hypothesis is that Smad4 positively regulates NK cell development and cytotoxicity against target cells including oHSV-infected GBM cells, and this can be modulated by temporarily inhibiting BMP-Smad4 signaling to enhance the efficacy of oHSV therapy. We propose an in-depth investigation into the molecular mechanisms whereby smad4 positively regulates Gzmb expression, cytotoxicity, and development of NK cells in mice using our aforementioned animal model and in humans using samples of patients with familial juvenile polyposis (FJP), having a germline loss-of-function mutation in Smad4. Additionally, we also propose to modulate BMP- Smad4 signaling to temporarily inhibit NK cell responses to oHSV to enhance its efficacy for the treatment of GBM. These are outlined in three Aims to test our hypothesis. Aim 1 is to dissect the mechanisms by which Smad4 positively regulates NK cell cytotoxicity against target cells. Aim 2 is to characterize the mechanisms by which Smad4 positively regulates NK cell development. Aim 3 is to temporarily inhibit BMP-Smad4 signaling in NK cells to improve oncolytic viral therapy for GBM in vitro and in vivo. We believe that the results of these studies will lend new insights into basic mechanisms of cytotoxic cell therapy and that exploration of novel therapeutics in preclinical models will advance the treatment of GBM.